Nucleic acid nanostructure barcode probes

ABSTRACT

Provided herein are, inter alia, barcode probes comprised of transiently or stably fhiorescently labeled nucleic acid nanostructures that are fully addressable and able to be read using standard fluorescent microscope and methods of use thereof including methods of use as detectable labels for probes.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/408358, filed on Oct. 29, 2010, entitled “DNA NANOSTRUCTUREBARCODE PROBES”, the entire contents of which are incorporated byreference herein.

REFERENCE TO A COMPACT DISK APPENDIX

A computer program listing is appended to this disclosure and is herebyincorporated herein by this reference. The appendix includes the fileprogramlisting.txt which includes listings for 6ht-v5-1color-1.json and6ht-v6-2color-1.json.

BACKGROUND OF INVENTION

A major challenge in modern biology and nanotechnology is to observe,identify and differentiate a large number of different molecular speciesin real time. Fluorescent microscopy is a powerful tool fornon-destructively and dynamically monitoring many individual molecularevents. However, the multiplexing ability of fluorescent imaging islimited by the number of spectrally non-overlapping fluorophoresavailable. There is therefore a great need for novel addressablefluorescent probes useful in multiplex detection systems.

BRIEF SUMMARY OF INVENTION

The invention provides, inter alia, nucleic acid barcode probes thatcomprise a nucleic acid nanostructure comprising one or morefluorescently labeled regions that may be stably or transiently bound(and thus labeled) with fluorophore-bearing oligonucleotides. Thebarcode probes may further comprise a target binding moiety. Theinvention also provides methods of use for such barcode probes includingbut not limited to their use in analyte detection assays (e.g., assaysfor detecting and optionally quantitating one or more analytes). Thenature of the fluorescent signals (e.g., the wavelength or “color”,intensity, etc.) and the pattern (or orientation, or arrangement orgeometry) of such signals on the barcode can also be used to identifyparticular analytes in a sample. This facilitates multiplexed assays inwhich a plurality of analytes are detected simultaneously (or at aminimum with a single sample or a single aliquot from a sample).

In one aspect, the invention provides a method of detecting a targetcomprising: contacting a sample with a nucleic acid barcode probe, anddetermining whether the nucleic acid barcode probe binds to one or morecomponents in the sample, wherein binding of the nucleic acid barcodeprobe to one or more components of the sample indicates presence of atarget in the sample, and wherein the nucleic acid barcode probecomprises a nucleic acid nanostructure comprising a target bindingmoiety and at least two fluorescently labeled regions.

In some embodiments, the method further comprises identifying the targetbased on the color and/or orientation of the fluorescently labeledregions of the nucleic acid barcode probe bound to one or morecomponents of the sample.

In some embodiments, whether the nucleic acid barcode probe binds to oneor more components in the sample comprises contacting the nucleic acidbarcode probe with soluble, transiently binding fluorophore-bearingoligonucleotides.

In some embodiments, the nucleic acid barcode probe comprises stablybound fluorophore-bearing oligonucleotides.

In another aspect, the invention provides a method of detecting a targetcomprising contacting the target with a nucleic acid barcode probe,under conditions sufficient for the target to bind to the nucleic acidbarcode probe; separating the target from material that is not bound tothe target; and detecting the presence of the nucleic acid barcode probebound to the target, wherein the nucleic acid barcode probe comprises anucleic acid nanostructure comprising a target binding moiety and atleast two fluorescently labeled regions.

In some embodiments, the method further comprises identifying the targetbased on the color and/or orientation of the fluorescently labeledregions of the nucleic acid barcode probe bound to the target.

In some embodiments, detecting the presence of the nucleic acid barcodeprobe bound to the target comprises contacting the nucleic acid barcodeprobe with soluble, transiently bound fluorophore-bearingoligonucleotides.

In some embodiments, the nucleic acid barcode probe comprises stablybound fluorophore-bearing oligonucleotides.

In another aspect, the invention provides a nucleic acid barcode probecomprising a nucleic acid nanostructure, such as a DNA barcode probecomprising a DNA nanostructure, having at least twofluorescently-labeled regions. In some embodiments, the nanostructurecomprises at least three fluorescently-labeled regions, and theorientation is determinable due to asymmetric spacing of thefluorescently-labeled regions. The locations of each of thefluorescently-labeled regions on the nanostructure are such that apattern of the fluorescent labels is determinable based on the emissionof visible light by the fluorescently-labeled regions. In someembodiments, the orientation of the barcode probe is determinable basedon the emission of visible light by the fluorescently-labeled regions.

In some embodiments, each of the fluorescently-labeled regions has acenter that is located at least 200 nm from the centers of the otherfluorescently-labeled regions. In some embodiments, each of thefluorescently-labeled regions has a center that is located at least 250nm from the centers of the other fluorescently-labeled regions. In someembodiments, each of the fluorescently-labeled regions has a center thatis located at least about 25 nm, at least about 50 nm, or at least about75 nm, or at least about 100 nm, or at least 150 nm from the centers ofthe other fluorescently-labeled regions.

In some embodiments, the nanostructure comprises a scaffold strand andplurality of staple strands. In some embodiments, the scaffold strandhas a sequence derived from M13 bacteriophage. In some embodiments, thefluorescently-labeled regions of the DNA nanostructure comprisefluorophore-labeled staple strands. In some embodiments, thefluorophore-labeled staple strand is a transiently bindingfluorophore-labeled staple strand. In some embodiments, thefluorophore-labeled staple strand is transiently binding at roomtemperature. In some embodiments, the fluorophore-labeled staple strandis 7-12 nucleotides in length. In some embodiments, thefluorophore-labeled staple strand is about 9 nucleotides in length. Insome embodiments, the fluorophore-labeled staple strand is a stablybinding fluorophore-labeled staple strand. In some embodiments, thefluorophore-labeled staple strand is a stably bindingfluorophore-labeled staple strand at room temperature. In someembodiments, the fluorophore-labeled staple strand is at least 18nucleotides in length, including in some embodiments 18-25 nucleotidesin length.

In some embodiments, the fluorophore-labeled staple strands are directlylabeled. In some embodiments, the fluorophore-labeled staple strands areindirectly labeled.

In some embodiments, the fluorophore-labeled staple strands have astaple domain hybridized to a scaffold strand and a handle domainhybridized to a fluorophore-labeled oligonucleotide. In someembodiments, the handle domain is 7-12 nucleotides in length. In someembodiments, the handle domain is about 9 nucleotides in length. In someembodiments, the handle domain is at least 18 nucleotides in length. Insome embodiments, the handle domain is 18-25 nucleotides in length.

In some embodiments, the nanostructure is a nanotube. In someembodiments, the nanostructure is prepared using a DNA origami method.In some embodiments, the nanostructure comprises a single-stranded DNAtile. In some embodiments, the nanostructure comprises DNA hairpins.

In some embodiments, the barcode probe further comprises a targetbinding moiety. In some embodiments, the target binding moiety is asingle-stranded nucleic acid complementary to a nucleic acid target. Insome embodiments, the target binding moiety is an antibody. In someembodiments, the target binding moiety is a protein or peptide.

In some embodiments, wherein the nanostructure, once assembled, issufficiently immutable in a resting state that the pattern of thefluorescent regions can be detected using light microscopy in thatresting state. In some embodiments, the resting state comprises absenceof an applied electric field.

In another aspect, the invention provides a composition comprising aplurality of any of the foregoing nucleic acid nanostructure barcodeprobes. In some embodiments, some or all members of the plurality aredifferent from other members in the plurality. The members may differfrom each other based on the target binding moiety and the signal andorientation (arrangements) of the barcode. In some embodiments, theplurality is equal to or less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, 200 or more. Insome instances, the plurality is equal to or less than 216.

In another aspect, the invention provides a method of identifying atarget nucleic acid comprising contacting the target nucleic acid withany of the foregoing barcode probes under conditions such that thetarget nucleic acid binds to the barcode probe; separating the targetnucleic acid from material that is not bound to the target nucleic acid;and detecting the presence of the barcode probe bound to the targetnucleic acid.

In another aspect, the invention provides a method of identifying atarget protein or cell comprising contacting the target protein or cellwith any of the foregoing barcode probes under conditions such that thetarget protein or cell binds to the barcode probe; separating the targetprotein or cell from material that is not bound to the target protein orcell; and detecting the presence of the barcode probe bound to thetarget protein or cell.

In another aspect, the invention provides a method of identifying atarget protein or cell comprising contacting the target protein or cellwith any of the foregoing barcode probes under conditions such that thetarget protein or cell binds to the barcode probe; separating the targetprotein or cell from material that is not bound to the target protein orcell; and detecting the presence of the barcode probe bound to thetarget protein or cell.

In another aspect, the invention provides a method of detecting a targetcomprising contacting a sample with any of the foregoing nucleic acidbarcode probes, and determining whether the nucleic acid barcode probebinds to one or more components in the sample, wherein binding of thenucleic acid barcode probe to one or more components in the sampleindicates presence of a target in the sample.

In some embodiments, the method further comprises removing components inthe sample that are not bound to the nucleic acid barcode probefollowing the contacting step.

In some embodiments, the target is a nucleic acid, a protein, a peptide,or a cell. or a combination thereof.

In some embodiments, the presence of the barcode probe is detected byfluorescent microscopy.

In some embodiments, the presence of the barcode probe is detectedwithout exposing the DNA barcode probe to an electric field.

In some embodiments, each probe comprises a nucleic acid nanostructurehaving at least two fluorescently-labeled regions and a target bindingmoiety, wherein the locations of each of the fluorescently-labeledregions on the nanostructure are such that a pattern of the fluorescentlabels of the barcode probe and the identity of the target bindingmoiety is determinable based on the emission of visible light by thefluorescently-labeled regions; and each of the fluorescently-labeledregions has a center that is located at least 200 nm from the centers ofthe other fluorescently-labeled regions.

In another aspect, the invention provides a population of nucleic acidbarcode probes such as DNA barcode probes, wherein each probe comprisesa nucleic acid nanostructure such as a DNA nanostructure having at leasttwo fluorescently-labeled regions and a target binding moiety, whereinthe locations of each of the fluorescently-labeled regions on thenucleic acid nanostructure are such that a pattern of the fluorescentlabels of the nucleic acid barcode probe and the identity of the targetbinding moiety is determinable based on the emission of visible light bythe fluorescently-labeled regions.

In some embodiments, each of the fluorescently-labeled regions has acenter that is located at least about 25 nm, at least about 50 nm, atleast about 100 nm, at least about 200 nm, or at least about 250 nm fromthe centers of the other fluorescently-labeled regions.

These and other aspects and embodiments of the invention will bedescribed in greater detail herein.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one Figure executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. Publication of the application includes gray-scalerenderings of the Figures. Color versions of these Figures are availableupon request.

FIG. 1. Design of the DNA nanotube based barcode. (A) Two schematicdrawings of the Blue--Red-Green (BRG, “--” and “-” denotes larger andsmaller inter-zone distance in the barcode, respectively) barcode with asegment diagram on the top and a 3D view at the bottom. The main-body ofthe barcode is a DNA nanotube formed from by dimerizing two origamimonomers, each consisting of 28 segments with 42-bp (13.6 nm) length.The grey segment in the middle represents the junction where the twomonomers are joined together through cross-hybridization between theirscaffolds and staples. Three 84-bp zones of the nanotube arefluorescently labeled (shown as blue, red and green segments) to producethe BRG barcode with an inter-zone distance of 450-nm between the firsttwo zones and 270-nm between the last two. Note that each zone is onlylabeled with one fluorophore species. The resulting barcodes are thusreferred as single-labeled-zone barcodes. (B) 3D cartoons showing thedetails of one fluorescently labeled zone. Left: a scaffold-plus-staplemodel of such an 84-bp zone before labeling. Each of the twelve63-base-long staples (shown in rainbow colors) contains two parts: the42-base region at the 5′-end weaves through three double-helices to foldthe scaffold (shown in black) into a six-helix bundle nanotube; and the21-base extension at the 3′-end protrudes out for fluorescent labeling.Middle: an identical but simplified model to emphasize the six-helixbundle structure (each helix shown as a semi-transparent grey cylinder)and the positioning of the twelve 21-base staple extensions (each shownas a light-grey curl). Right: Cartoon representation of a “green” 84-bpzone. The labeling is achieved by hybridizing the Cy3 (shown the glowinggreen spheres at the 3′-ends) modified strands to the staple extensions.

FIG. 2. Single-labeled-zone fluorescent barcodes. (A) Superimposed TIRFmicroscopy images of five barcode species (top) and the statistics frommanual counting (bottom). From left to right are the BBB, BRG, BRR, GRGand RGB barcodes with a representative image on top of the correspondingbar-graph. Each bar-graph is generated based on the manual sorting andcounting of the objects found in a 50×50 μm² image (˜40 barcodes, theexact sample size N is noted beside the corresponding bar-graph). (B) Arepresentative image of the equimolar mixture of 27 barcode species. (C)Statistics obtained by analyzing twenty-seven 50×50 μm² images of the 27barcode mixture (˜1,500 barcodes in total). Left: barcode counts of the27 species (average count of 55 with a standard deviation of 9). Arepresentative TIRF image (1.4×0.7 μm²) of each barcode type is placedunderneath the corresponding bar. Right: sorting result of the observedobjects shown as a pie-chart. Color scheme used for the bar-graphs andthe pie-chart (unrelated to the pseudo-colors of the fluorophores):blue: correct barcodes (qualified barcode with expected identity);green: incorrect barcodes (qualified barcode with unexpected identity);red: monomer nanotubes (one spot or two connecting spots); purple:barcodes with wrong geometry (i.e., bending angle)<120°; and orange:barcodes containing at least one spot with two colors. In the 27-barcodepool, correct vs. incorrect barcodes were not distinguishable becauseall barcode types are expected. As a result the bars and pierepresenting the qualified barcodes in (C) are shown in blue. Scalebars: 5 μm.

FIG. 3. Representative TIRF and software reconstructed images of BRG andGRG nano-barcodes. (A) TIRF image of BRG nano-barcodes. (B) Softwareconstructed image of (A), inset showing calculation of barcode bendingangle. (C) TIRF image of BRG nano-barcodes. (D) Software constructedimage of (C). (E) Theoretical designed distances between each region ofBRG (upper) and GRG (lower) nano-barcodes. Scale bar: 10 pixels or 0.714μm.

FIG. 4. Schematic illustration and result of a 21-mer oligonucleotidedetection using nano-barcodes. (A) A GRG barcode with a capture probe isbiotinylated as a result of the target binding and a nude BRG barcodestays untouched. Note that the drawing is not to scale. (B) TIRF imagesof nano-barcodes immobilized on streptavidin coated glass cover-slip.Left: with target; Right: without target. (C) Statistical detectionresult showing the average barcode counts per 71.4×71.4 μm² area. Errorbars represent the standard deviation.

FIG. 5. Dual-labeled-zone fluorescent barcodes. (A) Typical TIRFmicroscopy images of five selected barcode species, shown both inseparate channels and after superimposing. Scale bar: 5 μm. (B)Statistics obtained by analyzing two 50×50 μm² images of each barcodespecies (˜85 barcodes, the exact sample size N is noted beside thecorresponding bar-graph). The barcode types are noted under the x-axisof the diagram. Color scheme (unrelated to the pseudo-colors of thefluorophores): blue: correct barcodes (correct geometry and coloridentity); green: incorrect barcodes (correct geometry but incorrectcolor identity); red: monomer nanotubes (one spot or two connectingspots); and purple: barcodes with wrong geometry (i.e., bending angle<120⁰). (C) Computer-aided barcode counting results of the 72-barcodepool (N=2,617) and the 216-barcode pool (N=7,243) plotted as bar-graphswith descending barcode counts from left to right (data not shown). Acomputer-generated reference barcode image is placed underneath thecorresponding bar. (D) A table containing one representative TIRF image(1.4×0.7 μm²) for each of the 216 dual-labeled-zone barcode species.

FIG. 6. Super-resolution fluorescent barcodes. (A) Scheme of DNA-PAINTused for super-resolution barcode imaging. The 400 nm nanotube consistsof 4 binding zones spaced by ˜114 nm. Each zone can be decorated withthe desired combination of “docking” sequences for red, green or blueimager strands. The orthogonal imager strands bind transiently to theirrespective “docking” sites on the nanotube, creating the necessary“blinking” for super-resolution reconstruction (B) Top: Segment diagram(similar to the one used in FIG. 1) of the DNA nanotube monomers usedfor creating five barcodes for super-resolution imaging. Bottom:super-resolution images of the five barcodes shown in each channelseparately and as an overlay of all channels. Scale bar: 100 nm (C)Super-resolution image showing all five barcodes in one mixture. Scalebar: 500 nm.

FIG. 7. Tagging yeast cells with the GRG barcodes as in situ imagingprobes. (A) Cartoon illustrating the tagging mechanism. The biotinylatedbarcodes are anchored on the yeast cell through streptavidin moleculesbound to biotinylated polyclonal antibodies coated on the yeast surface.Only two of the ten biotinylated staples on the barcode are shown herefor clarity. (B) Overlaid microscope images (acquired in bright fieldand TIRF) of the yeast cells treated with the barcodes. Top: Yeast cellstreated as illustrated in (A). Bottom: Negative control: yeast cellstreated with non-biotinylated barcodes. Scale bars: 5 μm.

FIG. 8. Fluorescent barcode with non-linear geometry. (A) A schematic ofthree identical ˜400-nm long DNA nanotubes are linked to the outer edgeof a DNA ring with diameter of ˜60 nm through the hybridization betweenstaple extensions. The ring and the end of the tube are labeled by Cy3(green) and Cy5 (red), respectively. (B) A representative TIRFmicroscope image of the barcode shown in (a). Scale bar: 5 μm.

DETAILED DESCRIPTION OF INVENTION

Provided herein are nucleic acid (e.g., DNA) nanostructure barcodeprobes that are fully addressable and able to be read using standardfluorescent microscope.

Previously described molecular barcodes are constructed using one of twostrategies: (1) By combining multiple spectrally non-overlappingfluorophores in a controlled molar ratio to generate a mixed colorsignature; or (2) by separating fluorophores beyond the diffractionlimit (˜200 nm) and arranging them in a specific geometric pattern. Thefirst category of barcode has been made, for example, from dendrimericDNA (see, e.g., Li et al., Nat. Biotechnol. 23:885-889 (2005)),self-assembled 2D DNA arrays (see, e.g., Lin et al., Nano Lett.7:507-512 (2007)) and quantum dots (see, e.g., Han et al., Nat.Biotechnol. 19:631-635 (2001)). The successful construction and decodingof such barcodes heavily depends on both the fluorescent labelingefficiency and the imaging instrument's ability to precisely detectdifferent fluorescent intensity levels.

In contrast, barcodes constructed according to the second strategy aremore robust, and can be constructed and detected even when the labelingefficiency and imaging conditions are sub-optimal. Furthermore, whensuch methods are used, the ability to multiplex increases exponentiallywith each additional fluorescent spot that is incorporated. Examples ofbarcodes that fall in this category include rare-earth doped glassmicrofibers (˜100 μm long) (see, e.g., Dejneka et al., Proc. Nat. Acad.Sci. USA 100:389-393 (2003)) and double-stranded linear DNA with onestrand fluorescently tagged at specific locations (2-5 μm long) (see,e.g., Xiao et al., Nat. Methods 6:199-201 (2009) and Geiss et al., Nat.Biotechnol. 26:317-325 (2008)). However, existing methods using thisstrategy are limited both in the addressability of the fluorescent spotsand because, in the absence of specialized equipment, the labeledbarcodes will fold into a conformation that brings the fluorescent spotswithin the diffraction limit of visible light, rendering the barcodeunreadable using a fluorescent microscope.

The instant inventors recognized that the problems associated withexisting molecular barcode strategies could be addressed by usingnucleic acid nanostructures such as DNA nanostructures to create novelmolecular barcode probes. Nucleic acids such as DNA may be folded intopredetermined one-, two- or three-dimensional nanostructures using avariety of techniques, such as DNA origami (Rothemund US-2007/0117109A1), single-stranded tiles (Yin et al., “Programming DNA TubeCircumferences,” Science (2008): 321: 824-826), DNA hairpins (Yin et al.US-2009/0011956 A1; Yin et al., “Programming biomolecular self-assemblypathways,” Nature (2008) 451:318-323), and others.

In general, the DNA origami process involves the folding of one or morelong, “scaffold” DNA strands into a particular shape using a pluralityof rationally designed “staple” DNA strands. The sequences of the staplestrands are designed such that they hybridize to particular portions ofthe scaffold strands and, in doing so, force the scaffold strands into aparticular shape. Methods useful in the making of DNA origami structurescan be found, for example, in U.S. Pat. App. Pub. Nos. 2007/0117109,2008/0287668, 2010/0069621 and 2010/0216978, each of which isincorporated by reference in its entirety. Staple design can befacilitated using, for example, caDNAno software, available on theinternet at the cadnano website.

In certain embodiments, a DNA nanostructure barcode probe may include aDNA nanostructure made of one or more scaffold strands held in aspecific shape by rationally designed staple strands. The sequencelisting provides the nucleotide sequence of a scaffold strand, as SEQ IDNO:1, that may be used to construct an exemplary DNA origami barcodeprobe. The sequence listing also provides nucleotide sequences of staplestrands, as SEQ ID NOs: 13-361, that may be used to construct anexemplary DNA origami barcode probe, together with the scaffold strandrepresented by SEQ ID NO:1. As will be discussed in greater detailherein, SEQ ID NOs: 13-186 represent nucleotide sequences of staplestrands used to form a front monomer and SEQ ID NOs: 187-361 representnucleotide sequences of staple strands used to form a rear monomer in anexemplary DNA origami barcode probe. As used herein, the term “DNAnanostructures” is used for convenience and it is to be understood thatthe invention contemplates nucleic acid nanostructures generally. Thenanostructures of the invention may be linear (e.g., nanorods) ornon-linear (e.g., star-shaped, triangular, etc.).

The DNA nanostructure has at least two non-overlapping,fluorescently-labeled regions. In certain embodiments, the DNAnanostructure barcode probe has at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or12 non-overlapping, fluorescently-labeled regions.

The fluorophore pattern of the probe is determined using lightmicroscopy. In some embodiments, each of the fluorescently labeledregions can be visibly distinguished using a fluorescent lightmicroscope. In some of these embodiments, the center of eachfluorescently labeled region is separated from the centers of otherfluorescently labeled regions by a distance greater than the visiblelight diffraction limit of visible light (i.e. at least 200 nm). Incertain embodiments, therefore, each fluorescently labeled region is atleast 200 nm from any other fluorescently labeled region. In someembodiments, each fluorescently labeled region is at least 250 nm fromany other fluorescently labeled region.

In some embodiments, even higher resolution is achievable and thefluorescently labeled regions may be spaced apart from each other ateven shorter distances (i.e., a distance of less than 200 nm).Accordingly, in some embodiments, the distance between the fluorescentlylabeled regions may be equal to or about 175 nm, 150 nm, 125 nm, 100 nm,75 nm, 50 nm, 25 nm, or smaller distances. When the fluorescentlylabeled regions are spaced apart at distances that are less than thediffraction limit of visible light, the barcoded probes are imaged usingsuper-resolution techniques such as Point Accumulation for Imaging inNanoscale Topography (PAINT, DNA-PAINT), Stimulated Emission DepletionMicroscopy (STED), Reversible Saturable Optical Fluorescence Transitions(RESOLFT), Stochastic Optical Reconstruction Microscopy (STORM, dSTORM),Photoactivated Localization Microscopy (PALM), Blink Microscopy (BM),and any other form of super-resolution microscopy.

It is to be understood that the term “fluorescently-labeled region”embraces regions that are stably labeled and those that are transientlylabeled with fluorophore-bearing moieties such as fluorophore-bearingoligonucleotides that are complementary to a handle domain or dockingstrand (or sequence), as described in greater detail herein. In someinstances, the barcode probes of the invention are provided with suchfluorophore-bearing oligonucleotides bound thereto, and therefore theseprobes would be fluorescent. In other instances, the barcode probes ofthe invention are provided without such fluorophore-bearingoligonucleotides bound thereto, and therefore these probes may not befluorescent until they are bound to a fluorophore-bearingoligonucleotide. In these latter instances, a probe may be providedtogether with a plurality of fluorophore-bearing oligonucleotides thatare specific for (e.g., typically, complementary to) the handle domainsor docking strands of the probe. The probes and the oligonucleotides maybe provided in a kit, optionally with each in a separate containerwithin the kit.

The positions of the fluorescently labeled regions can be selected suchthat the orientation of the DNA nanostructure barcode may bedeterminable through visualization of the barcode on a fluorescentmicroscope (typically by some form of asymmetry in the arrangement ofthe labeled regions or by some arrangement convention, such a particularcolor or pattern reserved to indicate an end of the barcode). In someinstances, orientation information is unnecessary and the barcodepattern need not be absolutely unique. Thus, barcodes with even just twolabels may be used, and the presence and/or intensity of various labelsprovides the information required.

In some instances, the fluorescently labeled regions are arranged in alinear manner. An examples is shown in, for example, FIG. 1. Thefluorescently labeled regions may however be arranged in a non-linearmanner as well. An example is shown in FIG. 8. Other non-lineararrangements will be apparent to those of ordinary skill in the art andare embraced by the invention. In some instances, a non-linearorientation (or arrangement or geometry) of the fluorescently labeledregions is used to detect and/or identify analytes. In some instances,the signal (including for example the color or color combination) ofnon-linearly arranged fluorescently labeled regions is used to detectand/or identify analytes. In still other instances, orientation andsignal are used together to detect and/or identify analytes. Thus, it isto be understood that the nanostructure may be linear or non-linearand/or the arrangement of the fluorescently labeled regions may belinear or non-linear.

The DNA nanostructure, once assembled, is sufficiently immutable in aresting state (i.e., in the absence of external influence from e.g. anelectrical field) that the spatial pattern of the fluorescent regionscan be detected using light microscopy in that resting state. Portionsof the nanostructure may be flexible, such as unlabeled portions. Insome embodiments, a probe may include a (relatively) rigid portioncarrying label and an unlabeled flexible portion. in some embodiments, aprobe may have two rigid labeled regions connected by a deformablelinker.

In certain embodiments, the DNA nanostructure of the DNA barcode probecan be of any one-, two- or three dimensional shape that is able tosupport at least three fluorescently labeled regions at least 200 nmapart from each other. As stated herein, however, the invention is notso limited as nanostructures having fluorescently labeled regions spacedat distances of less than 200 nm from each other are also contemplated.

In certain embodiments, the DNA nanostructure is a nanotube. In someembodiments, the nanotube is at least 200, 300, 400, 500, 600, 700 or800 nm long. In certain embodiments, the DNA nanostructure is a sixhelix bundle dimer nanotube. Description of DNA nanotube design (via DNAorigami) can be found in, for example, Douglas et al., Proc. Natl. Acad.Sci. USA 104:6644-6648 (2006) and U.S. Pat. App. Pub. No. 2010/0216978,each of which is incorporated by reference in its entirety.

In some embodiments, the DNA barcode probes are designed such that theorientation of the DNA barcode probe is determinable based on theemission of visible light by the fluorescently-labeled regions. Anyproperty of the barcode can be used to render the orientation of the DNAbarcode can be determinable. For example, if the DNA barcode probe has asubstantially one-dimensional (linear) structure, such as a nanotube,the fluorescently labeled regions can be positioned asymmetrically alongthe DNA nanostructure. The orientation of a substantiallyone-dimensional (linear) DNA barcode probe can also be made determinableby asymmetrically using a particular fluorophore or combination offluorophores to label one side of the DNA nanostructure consistently. Ifthe DNA nanostructure is two- or three-dimensional, the orientation ofthe DNA barcode probe can also be rendered determinable based, forexample, on an asymmetry of the shape of the structure itself.

In certain embodiments, the fluorescently labeled regions of the DNAbarcode probe are generated by labeling specific staples of the DNAnanostructure with a fluorescent moiety. The staples can be directlylabeled or indirectly labeled. As used herein, the term “directlylabeled” refers to a nucleic acid that is covalently bonded to adetectable moiety. In contrast, the term “indirectly labeled” refers toa nucleic acid that is attached to a detectable moiety through one ormore non-covalent interactions.

In certain embodiments, certain staples of the DNA nanostructure aredirectly labeled with a fluorescent moiety. In such embodiments, thestaple can be, for example, synthesized with a particular fluorescentmoiety attached, or covalently bonded to a fluorescent moiety prior toits incorporation into the DNA nanostructure.

Any combination of fluorescent moieties may be used in a single barcode,provided the fluorescent signal from each labeled region is detectable.Preferably, the combination of fluorescent moieties is chosen so thatthere is no energy transfer between the fluorescent moieties (i.e., thefluorophore combination used on a single barcode probe does not containpairs of fluorophores that act together as a donor-acceptor pair).

In some embodiments, certain staples of the DNA nanostructure areindirectly labeled with a fluorescent moiety. In such embodiments, suchstaples can be synthesized to have at least two domains, a staple domainand a handle domain. The staple domain is a region (or nucleotidesequence) of the staple that hybridizes to the scaffold strand tocontribute to the formation and stability of the DNA nanostructure. Thehandle domain contains additional nucleic acid sequence that is notnecessary for the creation of the DNA nanostructure. Before, during orafter the formation of the DNA nanostructure, the handle sequences areavailable to be hybridized by oligonucleotides having a complementaryDNA sequence. Thus, such staples can be indirectly labeled byhybridizing the handle domain to another nucleic acid that has a nucleicacid sequence complementary to the handle and that is itself eitherdirectly or indirectly labeled with a fluorescent moiety.

In some embodiments, each fluorescent region of the DNA barcode probeincludes at least one fluorescently labeled staple. In certainembodiments, the fluorescently labeled region may include a plurality offluorescently labeled staples. For example, in certain embodiments, eachfluorescently labeled region may include at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or more fluorescently labeled staples. In regionsdesigned to have multiple staples labeled, all the staples may belabeled by a single type of fluorophore, or the staples may be labeledby a combination of fluorophore types. For example, in certainembodiments, a fluorescently labeled region is designed to be labeled bymultiple types of fluorophores present at a predetermined ratio. Anexample of this latter type of labeling, referred to as dual-labeling,is demonstrated in the Examples. In dual-labeling, a single region islabeled with two fluorophores in a predetermined ratio, to yield afluorescent signal that is distinguishable from either of thecontributing fluorophores. In this way, the variety of barcode probes isincreased.

In still other embodiments, the barcode probe may comprise one or moreregions that are transiently labeled with fluorophore-bearingoligonucleotide strands. These embodiments may be used when thefluorescently labeled regions are within 200 nm of each other. In someembodiments, all the regions contributing to the barcode will bedesigned to be transiently labeled. Transient labeling of a region isachieved by using a nanostructure comprised of staple strands withhandle domains that are shorter in length than those used for morepermanent binding of a fluorescently labeled oligonucleotide. As anexample, permanent (or stable) binding of fluorophore-bearingoligonucleotides to a handle domain of a staple strand can be achievedusing oligonucleotides and handle domains that are about 21 nucleotidesin length. When shorter oligonucleotides and handle domains are used,the strength of binding between the two is reduced and accordingly theyare more likely to dissociate than are longer strands. At roomtemperature, oligonucleotides and handle domains that are about 9nucleotides in length associate with each other only transiently. Aswill be understood in the art, at higher temperatures, the length of theoligonucleotide and handle domain will typically be increased in orderto achieve the same association/dissociation kinetics.

Accordingly, the invention contemplates handle domains that are directlyor indirectly labeled with fluorophores as well as those that aretransiently labeled with fluorophores. The length of the handle domain(and similarly its complementary oligonucleotide) will depend upon thenature of the binding (i.e., whether it is intended to be permanent ortransient binding), and the reaction conditions such as but not limitedto temperature, salt concentration, and the like. Such lengths mayrange, without limitation, from about 5 nucleotides to 30 nucleotides,or from about 7 nucleotides to about 25 nucleotides, or from about 9nucleotides to about 21 nucleotides.

In some embodiments, the DNA barcode probe may further include a targetbinding moiety that acts as a binding partner for the target (oranalyte) of interest. As used herein, the term “binding” refers to anassociation between at least two molecules due to, for example,electrostatic, hydrophobic, ionic and/or hydrogen-bond interactionsunder physiological conditions. Such a target binding moiety can be, forexample, without limitation, a nucleic acid such as an oligonucleotide,a protein or peptide such as an antibody or antibody fragment, acarbohydrate or a polysaccharide, or a small molecule. The targetbinding moiety and the target to which it binds can also be viewed in areceptor and ligand relationship. In certain embodiments, the DNAbarcode probe may include multiple target capture moieties, which may beidentical or different.

The target capture moiety can be attached to the DNA nanostructure ofthe DNA barcode probe using any method known in the art. For example,the target capture moiety can be covalently bonded to a staple strand ora scaffold strand. The capture moiety can also be indirectly attached toeither a staple strand or the scaffold strand by, for example,hybridizing to a handle domain of a staple strand, as described above.

In certain embodiments, the DNA barcode probe may include a targetbinding moiety that is an oligonucleotide or other nucleic acid capableof hybridizing to a target nucleic acid. In general, an oligonucleotideis capable of hybridizing to a target nucleic acid if it includes anucleic acid sequence that is substantially complementary a sequence ofthe target nucleic acid. In certain embodiments, the oligonucleotide mayinclude a sequence that is perfectly complementary to a target (i.e.that is able to base pair at every nucleotide with the target sequence).In some embodiments, the oligonucleotide is less than perfectlycomplementary to the target but is still able to hybridize to a targetnucleic acid under certain conditions. Thus, in certain embodiments thesequence of the oligonucleotide is at least 10%, 20%, 30%, 40%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% complementary to a sequence ofthe target nucleic acid. In certain embodiments, the DNA barcode probemay include a target binding moiety that is an aptamer.

In certain embodiments, the DNA barcode probe may include a targetbinding moiety that is an antibody. As used herein, the term “antibody”includes full-length antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chain thereof. The term “antibody”includes, but is not limited to, a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, or an antigen binding portion thereof. Antibodies may bepolyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; ormodified forms thereof (e.g., humanized, chimeric). As used herein, thephrase “antigen-binding portion” of an antibody, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. The antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of theV_(H), V_(L), CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and CH1domains; (iv) a Fv fragment consisting of the V_(H) and V_(L) domains ofa single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544 546), which consists of a V_(H) domain; and (vi) anisolated complementarity determining region (CDR) or (vii) a combinationof two or more isolated CDRs which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, V_(H) and V_(L), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(H) and V_(L)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423 426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

In certain embodiments the target binding moiety is a protein orpeptide. For example, a protein or peptide can be used to bind the DNAbarcode probe to the protein's ligand. DNA barcodes having protein orpeptide target binding moieties can also be used, for example, toidentify antibodies or receptors such as but not limited to cell surfacereceptors such as but not limited to T cell receptors and B cellreceptors and intracellular receptors such as but not limited to hormonereceptors that are able to bind to an epitope present on the protein orpeptide.

In certain embodiments, the target binding moiety is a small molecule.DNA barcode probes having small molecule target binding moieties can,for example, be used to identify molecular or cellular targets of smallmolecules of interest. Furthermore, libraries of small molecules, whereeach small molecule is individually tagged with a DNA barcode, can beused in small molecule library screens to identify small molecules thatspecifically bind to a target of interest.

DNA barcode probes may be provided as a population of distinct species.The number of distinct barcode probes in the population is limited onlyby the multiplexing capability of the particular barcodes in thepopulation. In certain embodiments, the population contains at least 10.50, 100, 500, 1000, 2000, 3000, 4000. 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ distinct barcode probes. The population maycontain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more distinctspecies. The population may contain less than or equal to about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100or more distinct species. In some embodiments, the population comprises216 distinct barcode probes. The barcode probes described herein aresufficiently adaptable and expandable to permit creation of suchlarge-scale populations of distinguishable species.

Methods of Use

The DNA nanostructure barcode probes can be used, inter alia, in anyassay in which existing molecular barcode technologies are used. Forexample, the DNA barcode probes can be used according to the methodsdescribed in U.S. Pat. App. Pub. No. 2010/0015607, the content of whichis incorporated by reference in its entirety.

Typically assays include detection assays including diagnostic assays,prognostic assays, patient monitoring assays, screening assays,biowarfare assays, forensic analysis assays, prenatal genomic diagnosticassays, and the like. The assay may be an in vitro assay or an in vivoassay. The sample being analyzed may be a biological sample, such asblood, sputum, lymph, mucous, stool, urine and the like. The sample maybe an environmental sample such as a water sample, an air sample, a foodsample, and the like. The assay may be carried out with one or morecomponents of the binding reaction immobilized. Thus, the targets or thebarcodes may be immobilized. The assay may be carried out with one ormore components of the binding reaction non-immobilized. The assays mayinvolve detection of a number of targets in a sample, essentially at thesame time, in view of the multiplexing potential offered by the barcodeprobes of the invention. As an example, an assay may be used to detect aparticular cell type (e.g., based on a specific cell surface receptor)and a particular genetic mutation in that particular cell type. In thisway, an end user may be able to determine how many cells of a particulartype carry the mutation of interest, as an example.

In certain embodiments, a method of identifying a target nucleic acidmay include contacting the target nucleic acid with a DNA barcode havingan oligonucleotide target capture moiety that includes a nucleic acidsequence capable of hybridizing to a sequence of the target nucleicacid. In certain embodiments, the target nucleic acid is contacted witha DNA barcode probe under conditions such that the target nucleic acidbinds to the DNA barcode probe. In some embodiments, the target nucleicacid is then separated from at least some material that is not bound tothe target nucleic acid. In certain embodiments the presence of thetarget nucleic acid is then determined by detecting the presence of theDNA barcode probe bound to the target nucleic acid. The detection of theDNA barcode probe can be accomplished by any appropriate method known inthe art, including through fluorescent microscopy.

In some embodiments, the above-described method may further includeimmobilizing a capture probe containing a nucleic acid sequencecomplementary to a second target nucleic acid sequence to a solidsupport. In some embodiments the target nucleic acid is contacted withboth the capture probe and the DNA barcode probe. The target nucleicacid can be contacted by the capture probe first, the DNA barcode probefirst, or by both probes simultaneously. The capture probe can beimmobilized on the solid support before hybridizing to the targetnucleic acid, after hybridizing to the target nucleic acid, orsimultaneously with hybridizing to the target nucleic acid. In suchembodiments, the presence of the target nucleic acid will cause theformation of a tertiary complex that includes the capture probe, thetarget nucleic acid and the DNA barcode probe, immobilized to a solidsupport. As described above, the presence of the target nucleic acid isthen determined by detecting the presence of the DNA barcode probe byany appropriate method known in the art, including through fluorescentmicroscopy

In certain embodiments, a method of identifying a target protein or cellmay include contacting the target protein or cell with a DNA barcodeprobe described herein. In certain embodiments, the DNA probe has aprotein, peptide or antibody target capture moiety. In some embodiments,the DNA barcode probe is contacted to a target cell or protein underconditions such that the target cell or protein binds to the DNA barcodeprobe. In some embodiments, the method further includes the step ofseparating the target protein or cell from at least some material thatis not bound to the target protein or cell. In certain embodiments, themethod further includes identifying a target protein or cell bydetecting the presence of the DNA barcode probe bound to the targetprotein or cell. In some embodiments, the target protein is an antibodyor receptor such as but not limited to a T cell receptor.

It will be appreciated that for some of the aspects and embodiments ofthe invention, it is preferable for the barcode probe and/or target toremain stationary during image acquisition, particularly if asuper-resolution approach is being used and/or if the imaging techniquerequires superimposition of separately acquired images.

Super-Resolution Imaging

As discussed herein, in some aspects of the invention, the barcodeprobes comprise fluorescently labeled regions that are spaced atdistances less than 200 nm (i.e., the diffraction limit of visiblelight). In some of these instances, the probe is contacted to a sampleand once bound to one or more components of that sample it is exposed tosoluble oligonucleotides each of which is specific for one of theregions and is labeled with fluorophores. Such soluble, transientlybinding oligonucleotides may be referred to herein as “imager” strands.In some instances, the unbound components in the sample are removedprior to the addition of the fluorophore-bearing oligonucleotides. Thepresence of the barcode probe (and its identity based on its barcode) isthen determined by imaging the remaining sample components using aseries of single time lapsed images or a time lapsed movie in order todetect the binding of a fluorophore-bearing oligonucleotide to itscomplement on the probe. The complement on the probe may be referred toherein as a “docking” strand. The conditions are set such that at anygiven time only a single fluorophore-bearing oligonucleotide is bound tothe barcode probe. If only a single fluorophore is bound at any giventime, its color identity can be determined even if the neighboringregions are less than 200 nm away (since there would be no fluorophoresbound to the neighboring regions at the same time). In this way, one isable to position adjacent “fluorescently labeled” regions at distancesof less than 200 nm, and thereby generate an even greater diversity ofbarcodes. It is to be understood that in these embodiments, thefluorescently labeled regions are only transiently fluorescent and thatthe barcodes are only fluorescent when bound by imager strands.

In some embodiments, the imager and/or docking strands are about 5 toabout 18 nucleotides in length, or about 6 to about 15 nucleotides inlength, or about 7 to about 12 nucleotides in length. In someembodiments, the length is about 8, 9, 10, 11 or 12 nucleotides.

The art is familiar with other localization-based super-resolutionfluorescence techniques including, for example, the STORM technique asdescribed in U.S. Pat. No. 7,838,302, the disclosure of which relatingto STORM is incorporated by reference herein. The invention contemplateslabeling of the nanostructure barcode probes in any manner sufficientfor any super-resolution technique.

In other aspects of the invention, the nanostructure barcode probes ofthe invention may be used to calibrate and/or confirm calibration offluorescence microscopy equipment, including standard or conventionalfluorescence microscopes, confocal microscopes, super-resolutionfluorescence microscopes, and the like. Accordingly, nanostructurebarcode probes may be used as calibration standards (or tools). Ofparticular interest in these instances are the barcode probes having“fluorescently-labeled” regions that are spaced apart at less than thediffraction limit of visible light (i.e., less than about 200 nm). Whenused in this manner, the probes may or may not have a target bindingmoiety. As an example, they may comprise a target binding moiety such asbiotin or streptavidin that facilitates their immobilization for imagingpurposes.

EXAMPLES Example 1 Creation of a DNA Barcode

DNA origami barcode probes were constructed by fluorescently labelingthree separate regions of an 800-nm six-helix bundle dimer nanotube(FIG. 1). Each of the approximately 28 nm long fluorescently labeledregions was modified with twelve oligonucleotides attached to one of thethree fluorophores (rhodamine-green, Cy3 or Cy5). The DNA origamibarcode probe was designed to be asymmetric by placing the first twolabeled regions further apart (approximately 460 nm apart) than the lasttwo (approximately 270 nm apart). It is to be understood that otherarrangements, including symmetric arrangements, are also contemplated bythe invention.

The main structure of the DNA origami barcode probe is a DNA six-helixbundle nanotube dimer designed using the caDNAno software available atthe cadnano website. The sequence of the scaffold strand used for eachmonomer is provided as SEQ ID NO: 1. The sequences of the staples usedin the construction of the DNA origami barcode are provided as SEQ IDNOs: 13-361. Certain selected staples were elongated (after rationaldesign) to include 21-nt single stranded overhangs (handles) forfluorescent labeling. Table 1 provides the handle and anti-handlesequences used.

TABLE 1  DNA sequences used for fluorescently labeling nano-barcode.Name Sequence (SEQ. ID. NO.) Handle 1 5′-TTCCTCTACCACCTACATCAC-3′(SEQ ID NO: 2) Handle 2 5′-TAACATTCCTAACTTCTCATA-3′ (SEQ ID NO: 3)“Blue” 5′-GTGATGTAGGTGGTAGAGGAA/rhodamine green/-3′ anti-handle 1(SEQ ID NO: 4) “Green” 5′-GTGATGTAGGTGGTAGAGGAATTT/Cy3/-3′ anti-handle 1(SEQ ID NO: 5) “Red” 5′-GTGATGTAGGTGGTAGAGGAA/Cy5/-3′ anti-handle 1(SEQ ID NO: 6) “Blue” 5′-TATGAGAAGTTAGGAATGTTA/Alexa Fluor 488/-3′anti-handle 2 (SEQ ID NO: 7) “Green” 5′-TATGAGAAGTTAGGAATGTTA/Cy3/-3′anti-handle 2 (SEQ ID NO: 8) “Red” 5′-TATGAGAAGTTAGGAATGTTA/Cy5/-3′anti-handle 2 (SEQ ID NO: 9)

Accordingly, of the staple strands provided in the Sequence Listing, SEQID NOs: 16, 17, 70-73, 126-129, 182 and 183 are labeled “blue”; SEQ IDNOs: 195, 196, 239, 240, 251, 252, 295, 296, 307, 308, 351 and 352 arelabeled “red”; and SEQ ID NOs: 216-219, 272-275 and 328-331 are labeled“green”.

caDNAno JSON files showing the arrangement of the front and rearmonomers of the probe are provided in the computer program listingappendix as “6ht-v5-1color-1.json” and “6ht-v6-2color-1.json”respectively.

To assemble the DNA origami barcode probe, the front and rear monomerswere each assembled and fluorescently labeled in a separate test tubeand then mixed together to form the dimer. The assembly of each monomeris accomplished in one-pot reaction by mixing 100 nM scaffold strandderived from M13 bacteriophage (termed p7308) (SEQ ID NO:1) with a poolof oligonucleotide staple strands (SEQ ID NOs: 13-186 for the frontmonomer and 187-361 for the rear monomer; 600 nM of each; reverse-phasecartridge purified, Bioneer Inc.) in folding buffer containing 5 mMTris, 1 mM EDTA, 20 mM MgCl₂, 50 mM NaCl (pH 8) and subjecting themixture to a thermal-annealing ramp that cooled from 80° C. to 60° C.over the course of 80 minutes and then cooled from 60° C. to 24° C. over15 hours. Excessive staples were removed by polyethylene glycol (PEG)fractionation before each monomer was incubated with anti-handlescarrying appropriate fluorophores at 1:1.2 molar ratio for labeling. Fordimerization, a stoichiometric amount of the fluorescently labeled frontand rear monomers were mixed and incubated a 37° C. for 2 hours. Thefinal product was purified by agarose gel (non-denaturing, 1.0%)electrophoresis. The purified nano-barcode can be stored at −20 ° C. forat least 3 months.

Although this example uses a barcode made of two monomers, a DNA barcodeprobe could include 3, 4, 5, or more monomers. The platform is thereforescalable to any desired level, and can provide any desired number ofdistinct probes.

Example 2 Detection of a Singly-Labeled DNA Barcode Using FluorescentMicroscopy

Two of the monomer nanotubes were folded and fluorescently labeledseparately and combined together as described above to yield thenano-barcode. The final product was purified by agarose gelelectrophoresis and directly deposited on a glass slide for imagingusing a total internal reflection fluorescence microscope (TIRFM). Inbrief, 5 μL of the purified nano-barcode (˜20 pM) was deposited on aglass slide, sandwiched by a coverslip (No. 1.5, 18×18 mm², ˜0.17 mmthick) and let sit for 5 minutes before being imaged on a Leica DM16000BTIRFM. The samples were imaged sequentially through three channels, eachassigned a false color (blue, green and red). For the blue channel, the488 nm laser beam was reflected by a dichroic mirror (430/505/575/670)and shined on the sample through a 100× objective (HCX PL APO 100×/1.47oil CORR TIRF, Leica). The emission light was collected through the sameobjective, filtered through the same dichroic mirror and an externalemission filter (525/36) and integrated for 800 ms on an EM-CCD camera(Hamamatsu C9100-02). For the green and red channels, similarconfigurations were used except for the excitation laser (561 and 635nm, respectively), emission filter (605/52 and 705/72, respectively) andexposure time (500 and 700 ms, respectively). The imaging process wasautomatically controlled by a Leica LAS software. The images wereprocessed by Image J and a custom written software for decoding.

The following false-colors were assigned to the fluorescently labeledregions: Alexa Fluor 488/rhodamine green (blue), Cy3 (green) and Cy5(red). TIRFM images resolved the DNA origami barcode as a strip of threedistinct bright spots. Notably, the distance between blue and greenspots is farther than that between green and red showing the asymmetryof the barcode (data not shown)

A DNA origami barcode system having three fluorescently labeled regions,each of which is labeled by one of three distinct fluorophores cangenerate 3³ (27) distinct DNA origami barcode probes. All 27 possibleDNA origami barcode probes were assembled separately and purifiedtogether by agarose gel electrophoresis.

Five distinct barcodes from the 27 members in the barcode family wererandomly chosen for quality control experiments. The barcodes wereassembled and purified separately and imaged under the same experimentalconditions. Two distinct features of the barcode were clearly visiblefrom the TIRF images (FIG. 2A, top panel): first, each fluorescentlylabeled zone on a barcode was resolved as a single-color spot and eachcomplete barcode consisted of three of such spots; Second, two of theneighboring spots were separated by a small gap while the other 7 twoneighbors sat closely together. Therefore one can visually recognize anddecode those geometrically encoded barcodes based on the color identityof the spots and their relative spatial positions, even without the aidof any specialized decoding software. Using a custom-written softwarethat localizes the center of each spot on the BRG barcodes, we measuredthe average center-to-center distance between the neighboring spots tobe 433±53 nm (mean±s.d., N=70; larger distance) and 264±52 nm(mean±s.d., N=70; smaller distance), confirming the correct formation ofthe barcodes. These experimentally measured distances were slightlysmaller than the designed values (478 nm and 298 nm). We attribute thisdiscrepancy to random thermal bending of the nanotubes (persistencelength of ˜1-2 μm), which has been observed previously by others²³²⁸ andconfirmed by us (data not shown) using transmission electron microscopy(TEM). It is important to note that unlike some other geometricallyencoded barcoding systems (e.g., NanoString nCounter¹²), there was nomolecular combing step involved in the sample preparation. Theseparation between the fluorescent spots was exclusively created by theinherently rigid structure of the six-helix bundle nanotube. It is alsonotable that the spot intensities were not perfectly uniform across thewhole image, which can be explained by factors such as the unevenillumination of the sample stage and differences in labeling efficiency.Nevertheless, the TIRF images proved that the barcodes were successfullyassembled and can be resolved unambiguously. We then manuallyinvestigated TIRF images with an area of 50×50 μm² for the selected fivebarcodes (FIG. 2A, bottom panel). The objects found within the imageswere first sorted into qualified (i.e., three single-color spotsarranged in a nearly linear and asymmetric fashion as designed) andunqualified (i.e., all other objects) barcodes. The qualified 8 barcodeswere further categorized into correct and incorrect (false-positive)barcodes based on the fluorescent signatures of the composing spots toreflect whether the barcode was the expected type. The unqualifiedbarcodes were further sorted into (1) monomer nanotubes (single spot ortwo “kissing” spots), (2) barcode with “wrong” geometry (i.e., extremebending), and (3) barcode containing at least one spot with multiplecolors. Our statistics revealed that more than 70% of the visibleobjects were qualified barcodes, which we further determined to beexclusively the expected type (i.e., zero false-positive out of 188qualified barcodes observed). The unqualified barcodes arose likely fromfolding defects, sample damage during handling and overlapping nanotubeson the surface, which can be largely reduced by optimizing the samplepreparation and imaging protocol.

Typically, a number of different barcode species coexist in one pool.Thus it is important to examine the robustness of our system by mixingdifferent types of barcode together. In an initial test, we synthesizedBRG and RGB barcodes separately, mixed them together at equal molarratio and co-purified them via gel electrophoresis. The TIRF analysis ofthe purified mixture confirmed the 1:1 stoichiometry of the two barcodesand the overall assembly success rate (qualified barcode/all objects) of˜80%, suggesting that both barcodes maintained their integrity in themixing and co-purification process. In addition, over 98% of thequalified barcodes fell into one of the two expected types (BRG andRGB). The 2% false-positive rate was due to an unexpected barcode namelyBGB, which could be attributed to a rare occasion when the front monomerof the BRG barcode lay in proximity of the rear monomer of the RGBbarcode. This can be eliminated using a more stringent purificationcondition to minimize the amount of leftover monomers.

We next challenged the system by imaging a pool of all 27 members of thebarcode family in which all species were mixed at equimolar amount. TheTIRF images (FIG. 2B) showed that all types of barcodes were resolved.Statistics by sampling twenty-seven 50×50 μm² images (1,500 barcodes intotal) revealed an average count of 55 per barcode type with a standarddeviation of 9 (FIG. 2C), fitting well with the expected stoichiometryconsidering the pipetting and sampling error. The distribution ofobserved objects over the four categories (note that here correct vs.incorrect barcodes were not distinguishable as all 27 types wereincluded) was consistent with the values measured from the single-typebarcode samples. The above observations suggest that thesub-micrometer-long DNA nanotube represents a reliable platform toconstruct geometrically encoded barcodes with built-in structuralrigidity.

Software could be used to recognize and decode the image of differenttypes of DNA origami barcode probes automatically. Typically, suchsoftware has three basic steps: recognition, filtering and decoding. Inthe first step, the fluorescent spots are identified. For eachfluorescent channel (blue, red or green), the software identifiesfluorescent spots based on their intensity and size, then compares themto automatically determined thresholds using overall image intensity andpre-defined spot sizes. The fluorescent spots below those thresholds areremoved along with general background noise. After processing all threechannels, a barcode image is constructed by merging spots identified inthree channels. An example of such a barcode image is provided in FIG.3. In the second step, the distances (calculated from (x,y) coordinatesof fluorescent spots) between each region of DNA origami barcode probesand the barcode bending angle (based on the center spot) are calculated.The software then filters DNA origami barcode probes and removes thosewith region distances that disagreed with the theoretical regiondistances or had a barcode bending angle smaller than a currentlyarbitrarily set threshold angle of 140 degrees. As an example, the BRGbarcodes were filtered using three distance rules: (1) The distancebetween blue and green spot cannot be greater than 15 pixels(theoretical distance was 11 pixels); (2) The distance between red andblue spot cannot be greater than 9 pixels (theoretical distance was 6.8pixels); and (3) The distance between red and green spot cannot begreater than 6 pixels (theoretical distance was 4.2 pixels). The BRGbarcodes that had a bending angle (at the red spot) smaller than 140degrees were removed. In the last step, after all barcodes areidentified and filtered, the software uses different criteria (based ontheoretical distance and color) to sort barcodes into specific types.Therefore, multiple types of DNA barcode probes can be decoded based onhow they were originally designed. Besides the final constructed DNAbarcode probe image, the software can output statistical informationregarding the (x,y) coordinates of each DNA barcode probe fluorescentspot, distances between adjacent spots, barcode bending angle, andbarcode types.

The software described above was used to detect BRG and GRG DNA origamibarcode probes. As shown in FIG. 3, the BRG and GRG DNA origami barcodeprobes were clearly identified by the automated decoding software. Thesoftware used a number of filters to select the valid DNA origamibarcode probes. As a result, 88% and 85% of BRG and GRG nano-barcodeswere counted. The software generated statistical information on BRG andGRG barcodes (Table 2) that showed that the measured distances betweeneach region on those DNA origami barcode probes were in good agreementwith the theoretical region distances as designed (FIG. 3E).

TABLE 2 Bar- Barcode Barcode Barcode code identi- dis- bending Distance1 Distance 2 type fied carded angle α (degree) (pixel) (pixel) BRG 70 9165.11 ± 10.08 6.18 ± 0.76 3.77 ± 0.74 GRG 81 15 161.51 ± 11.73 6.12 ±0.95 3.95 ± 0.98

Example 3 Detection of an Oligonucleotide Target Using a DNA BarcodeProbe

A 42-nt DNA oligonucleotide target was detected using a DNA origamibarcode probe. Two different purified barcodes Green, Red, Green (GRG)with the 21-nt oligonucleotide target capture moiety specific for a42-nt oligonucleotide target (Target 1, see Table 3 for sequences) andBlue, Red, Green (BRG) without a target capture moiety, were mixed at1:1 ratio and diluted to 20 pM in folding buffer. Biotin probe specificfor Target 1 (0.2 μL of 100 nM) and Target 1 (0.4 μL, of 10 nM) wasadded to 100 μL of the barcode mixture. In a separate test tubecontaining 100 μL of the barcode mixture, 0.2 μL of 100 nM biotin probefor Target 1 and 0.4 4. of 1× folding buffer was added as a negativecontrol. The above samples were both incubated at 37° C. overnightbefore being run through separate channels of a micro-fluidic cell witha streptavidin coated glass cover-slip (Xenopore) substrate. Bothchannels were then washed with 40 μL of washing buffer (5 mM Tris-HCl pH8.0, 500 mM NaCl) and imaged on a Leica TIRFM as described above. Tenimages were taken in each channel at random locations and the totalnumber of DNA origami barcode probes was counted manually.

TABLE 3  DNA sequences used in oligonucleotide target detection NameSequence (SEQ ID NO:) Target oligo5′-GAATCGGTCACAGTACAACCGCGCCGTAGGGCTG- ATCAAAGC-3′ (SEQ ID NO: 10)Biotin probe 5′-/Biotin/GCTTTGATCAGCCCTACGGCG-3′ (SEQ ID NO: 11) Capture5′-CGGTTGTACTGTGACCGATTC-3′ (SEQ ID NO: 12) probe

In the presence of the target oligonucleotide, the biotin probehybridized to one 21 nt sequence of the target oligonucleotide, whilethe GRG barcode hybridized to a second 21 nt sequence of the targetoligonucleotide. The BRG probe, however, lacking a target capture moietyspecific for the target oligonucleotide does not hybridize to the targetand therefore does not become bound by the biotin probe. (FIG. 4A). As aconsequence, when the detection reaction mixture was run through thestreptavidin-coated glass surface, the GRG barcode binds to the surface,while BRG barcode is washed away. TIRF images and statistical analysissupported the specificity of the detection (FIGS. 4B and C). In thepresence of 40 pM oligonucleotide target, there were ˜20 times more GRGbarcodes than BRG barcodes bound to the surface. In the absence of thetarget, both barcodes did not show significant surface binding.

Example 4 Dual-Labeled Barcode Probes

In order to enhance multiplexing capability even further, the sequenceof six staple extensions per zone was changed so that instead of usingtwelve identical fluorescent oligonucleotides for labeling, acombination of up to two fluorophores was used to create more uniquefluorescence signatures (pseudo-colors) for each zone. Six pseudo-colors(B, R, G, BG, BR, and GR) were generated by this “dual-labeling”strategy using three spectrally differentiable fluorophores.Consequently, the total number of distinct barcodes was raised from 27to 6³=216, which represented an order of magnitude increase in themultiplexing capability.

Similar to the single-labeled-zone barcode family, 5 members from thedual-labeled-zone barcode family were chosen for quality controlpurpose. The barcodes can be visually decoded either solely from thesuperimposed image or by examining all different channelssimultaneously. For example, as shown in the first column of FIG. 5A,the barcode “BG--GR-BR” (“--” and “-” denotes larger and smallerinter-zone distance in the barcode, respectively) exhibited two spotseach in the blue, green and red channels but with descending gapsbetween them, matching its design. In the superimposed image, thebarcodes were seen as Cyan--Yellow-Pink, an expected consequence ofcolor mixing caused by the dual-labeling strategy. In a similar fashion,we further verified the correct formation of the other four selectedbarcodes (FIG. 5A). Although the final pseudo-color from thedual-labeled zones was not always uniform (e.g., some yellow spots weregreen-tinted while the others were red-tinted) due to the inconsistentlabeling efficiency and minor sample displacement during imaging, thefluorescence signature of any given spot could be identified by checkingthe raw images acquired from the three imaging channels. We manuallyanalyzed two 50×50 μm² images of each dual-labeled-zone barcode andplotted the statistical data in FIG. 5B. Here, objects were sorted intoqualified barcodes and unqualified barcodes based on their geometry andthe qualified ones were further categorized as correct and incorrect.75-95% of the objects were qualified barcodes, among which 80-90% werethe correct type (percentage varies depending on the exact type ofbarcode). Compared to the single-labeled-zone barcode family, thepercentage of qualified barcodes remained the same, while the falsepositive rate increased significantly from zero to 10-20%. This observedincreased false positive rate is consistent with the expected decreasedrobustness of the dual-labeling strategy (as compared tosingle-labeling). In one design, a dual-labeled zone carried 6 stapleextensions for each fluorophore species, only half as many as in asingle-labeled zone.

As a result, the dual-labeled-zone barcode consisted of dimmer spotsthat were more susceptible to damages such as photo bleaching. In thissense, a single-labeled-zone barcode can be thought of as a redundantlyencoded dual-labeled-zone barcode. For instance, a single-labeled-zonebarcode can still be recognized as the correct type when sixfluorophores were missing from each zone, a scenario in which thedual-labeled-zone barcode could be disqualified or categorized asincorrect. In the latter case, it will increase the false positive rate.In principle, the false positive rate can be decreased by increasing thecopy number of each fluorophore species in a dual-labeled zone. The fivebarcodes we examined have each of their zones labeled with two distinctfluorophore species, making them likely among the most error-pronemembers of the dual-labeled-zone barcode family. Therefore, we wouldexpect a smaller false-positive rate on average from the whole family.

We further tested the dual-labeled-zone barcoding system by imaging amixture containing 72 barcode species that were individually assembledand co-purified. Custom MATLAB scripts were used to assist the decodingprocess in two steps. In step one, a three-channel (red, green, blue)TIRF image containing barcodes was pre-processed to remove backgroundand thresholded so that only pixels containing qualified barcodesremained. The resulting three-channel binary image was merged togenerate a single-channel binary image. Next, the software identifiedthe location and orientation of geometrically legitimate barcodes basedon their shape in the binary image. In step two, for each barcodelocated in step one, the corresponding region of the three-channel imagewas compared against a library of all possible reference barcodes. Theobserved barcode was assigned the identity of the reference barcode withthe highest correlation. The fully automated decoding process(unsupervised mode) ended after the above two steps. In an optionalsupervised mode, the software presented the user with the observedbarcode and its most likely identity for approval. Comparison betweensupervised and unsupervised decoding results confirmed >80% agreementbetween the computer and the user. The computer-aided (supervised mode)analysis of thirty-six 64×64 μm² three-channel images registered ˜2,600qualified barcodes that belonged to 116 different species (FIG. 5C, toppanel). The expected 72 species constituted ˜98% of the total barcodepopulation with an average barcode count of 36 per species and astandard deviation of 8. In contrast, the unexpected species averagedonly ˜1.4 barcodes per species (maximum 4 counts).

Finally, we analyzed a mixture containing all the 216 members of thedual-labeled-zone barcode. Sixty 64×64 μm² images of this mixture wereprocessed by the decoding software in the unsupervised mode. The fullyautomated analysis registered ˜34 barcode counts per species (7,200barcodes total) with a standard deviation of 17 (FIG. 5C, bottom panel).The relatively large standard deviation could be attributed to thedecoding error in the fully automated data analysis. Our studydemonstrated that 216 barcode species were successfully constructed andresolved (FIG. 5D).

Example 5 Super-Resolution Barcode Probes

Barcodes with higher spatial information density were also generatedusing geometrically encoded super-resolution barcodes with fluorescentfeatures spaced by ˜100-nm.

As a feasibility demonstration of the latter approach, we appliedDNA-PAINT³¹, a recently developed super-resolution fluorescencetechnique, to image the barcodes. Over the last years, severaltechniques have been developed that allow imaging beyond the diffractionlimit using far-field fluorescence microscopy⁴⁰⁻⁴⁴. In mostsuper-resolution implementations, fluorophores are switched betweenfluorescence ON- and OFF-states, so that individual molecules can belocalized consecutively. In methods relying on targeted readout schemessuch as in Stimulated Emission Depletion Microscopy⁴⁵ (STED) or otherReversible Saturable Optical Fluorescence Transitions⁴⁰ (RESOLFT)techniques, fluorescence emission is actively confined to an area belowthe diffraction limit Switching of fluorescent molecules can also becarried out stochastically such as in (direct) Stochastic OpticalReconstruction Microsco^(46,47) (STORM, dSTORM), PhotoactivatedLocalization Microscopy⁴⁸ (PALM) and Blink Microscopy⁴⁹ (BM) where mostfluorescent molecules are “prepared” in a dark state and onlystochastically switched on to emit fluorescence. In Point Accumulationfor Imaging in Nanoscale Topography⁵⁰ (PAINT), fluorescence switching isobtained by targeting a surface with fluorescent molecules. In allstochastic approaches, fluorescence from single molecules islocalized^(51,52) in a diffraction-limited area to yield super-resolvedimages. DNA-PAINT uses transient binding of fluorescently labeledoligonucleotides (imager strands) to complementary “docking” strands onDNA nanostructures to obtain switching between a fluorescence ON- andOFF-state, which is necessary for localization-based super-resolutionmicroscopy (cf. FIG. 6A). By adjusting the length of the imager/dockingstrand duplex and the concentration of imager strands in solution,fluorescence ON- and OFF-times can be tuned independently.

For this study, we extended the DNA-PAINT technique to three-colorimaging using orthogonal imager strand sequences coupled to threespectrally distinct dyes (Atto488 for blue, Cy3b for green and Atto655for red excitation). To demonstrate the feasibility of the three-colorsuper-resolution barcode system, we designed a DNA nanotube monomer with4 binding zones in a symmetric arrangement. The neighboring zones wereseparated by ˜114 nm (i.e., well below the diffraction limit) Eachbinding zone consists of 18 staple strands, which can be extended todisplay three groups of orthogonal sequences (six per group) for thered, green or blue imager strands to bind. As a proof-of-principleexperiment, we designed five different barcodes (FIG. 6A and top panelof B). The bottom panel of FIG. 6B shows the super-resolutionreconstruction of the five barcodes for each channel separately as wellas an overlay of all channels. FIG. 6C shows a larger area containingall five barcodes. The unique pattern of the barcodes in all threechannels can be resolved. Some barcodes moved during the sequentialimaging of all three color channels, but were still resolvable. Imagingcould be improved by alternating excitation and faster image acquisitionto prevent this effect. The transient, repetitive binding of imagerstrands to docking sequences on the nanotube not only creates thenecessary “blinking” behavior for localization but also makes theimaging protocol more robust, as DNA-PAINT is not prone tophoto-bleaching or incorrectly labeled strands. With the microscopesetup we used, DNA-PAINT provides a resolution of ˜46 nm (FWHM of aGaussian fit to the reconstructed PSF) in the red, ˜25 nm in the greenand ˜29 nm in the blue channel. The lower resolution in the red imagingchannel is a result from weaker laser excitation power. When using ahigher power TIRF system, a resolution of 24 nm, similar to the greenand blue channel can be obtained. The obtainable resolution and imagingspecificity suggests that 6 positions on one nanotube monomer could berobustly resolved while keeping the geometrical asymmetry of thebarcode, which would lead to 6⁷=279,936 possible different barcodes.Furthermore the modularity of the nanotube design enables the customizedreengineering of barcodes with inter-zone distances tailored to theresolving power of the used microscope, thus making it applicable for awide range of microscope setups.

Example 6 In Situ Labeling

In a proof-of-principle experiment, the GRG barcode was used to tagwild-type Candida albicans yeast. The yeast cells were first mixed witha biotinylated polyclonal antibody specific to C. albicans, then coatedwith a layer of streptavidin, and finally incubated with biotinylatedGRG barcodes (FIG. 7A). TIRF microscopy revealed the barcodes attachedto the bottom surface of the yeast cells (FIG. 7B, top panel). Whilesome of the nanotubes landed awkwardly on the uneven cell walls of theyeast cells, a number of GRG barcodes can be clearly visualized. Incontrast, no barcode tagging was observed when non-biotinylatedantibodies or barcodes were used to treat the yeasts (FIG. 7B, bottompanel), suggesting that little to no non-specific interaction existedbetween the barcode and the cell surface.

Example 7 Non-Linear Labeled Nanostructures

DNA nanostructures with non-linear geometry could be assembled togenerate more sophisticated barcodes. FIG. 8 shows an example wherethree ˜400 nm DNA tubes were linked to the outer edge of a ˜60 nm DNAring through hybridization between the staple extensions (FIG. 8A,inset). Fluorescently labeling the ring and the far end of the nanotubesgenerated a three-point-star-like structure clearly resolvable underfluorescence microscopy. TIRF microscopy and TEM studies (FIG. 8B)revealed that about 50% of successfully folded barcodes featured threenanotubes surrounding the ring with roughly 120° angle between eachother as designed, while many other barcodes had significantly biasedangles between neighboring nanotubes due to the semi-flexibledouble-stranded DNA linker between the ring and the nanotubes. It isconceivable that using similar design to connect three identical“satellite” linear barcodes to a central hub (here the three satellitebarcodes may share the hub as a common fluorescently labeled zone), onecan construct barcodes with triplicated encoding redundancy that featureoutstanding reliability. In addition, more rigid linkers between thering and the protrusions (e.g., multi-helix DNA with strand crossovers)could be employed to enforce better-defined barcode geometry.

REFERENCES

-   1 Han, M., Gao, X., Su, J. Z. & Nie, S. Quantum-dot-tagged    microbeads for multiplexed optical coding of biomolecules. Nature    biotechnology 19, 631-635, doi:10.1038/90228 (2001).-   2 Xu, H. et al. Multiplexed SNP genotyping using the Qbead system: a    quantum dot-encoded microsphere-based assay. Nucleic Acids Research    31, e43 (2003).-   3 Li, Y., Cu, Y. T. H. & Luo, D. Multiplexed detection of pathogen    DNA with DNA-based fluorescence nanobarcodes. Nature biotechnology    23, 885-889, doi:10.1038/nbt1106 (2005).-   4 Livet, J. et al. Transgenic strategies for combinatorial    expression of fluorescent proteins in the nervous system. Nature    450, 56-62, doi:10.1038/nature06293 (2007).-   5 Fournier Bidoz, S. et al. Facile and Rapid One-Step Mass    Preparation of Quantum-Dot Barcodes. Angewandte Chemie International    Edition 47, 5577-5581, doi:10.1002/anie.200800409 (2008).-   6 Lin, C., Liu, Y. & Yan, H. Self-assembled combinatorial encoding    nanoarrays for multiplexed biosensing. Nano letters 7. 507-512,    doi:10.1021/n1062998n (2007).-   7 Marcon, L. et al. ‘On-the-fly’ optical encoding of combinatorial    peptide libraries for profiling of protease specificity. Molecular    bioSystems 6, 225-233, doi:10.1039/b909087h (2010).-   8 Nicewarner-Pena, S. R. Submicrometer Metallic Barcodes. Science    294, 137-141, doi:10.1126/science.294.5540.137 (2001).-   9 Gudiksen, M. S., Lauhon, L. J., Wang, J., Smith, D. C. &    Lieber, C. M. Growth of nanowire superlattice structures for    nanoscale photonics and electronics. Nature 415, 617-620,    doi:10.1038/415617a (2002).-   10 Braeckmans, K. et al. Encoding microcarriers by spatial selective    photobleaching. Nature materials 2, 169-173, doi:10.1038/nmat828    (2003).-   11 Dejneka, M. J. et al. Rare earth-doped glass microbarcodes.    Proceedings of the National Academy of Sciences of the United States    of America 100, 389-393, doi:10.1073/pnas.0236044100 (2003).-   12 Geiss, G. K. et al. Direct multiplexed measurement of gene    expression with color-coded probe pairs. Nature biotechnology 26,    317-325. doi:10.1038/nbt1385 (2008).-   13 Pregibon, D. C., Toner, M. & Doyle, P. S. Multifunctional encoded    particles for high-throughput biomolecule analysis. Science 315,    1393-1396, doi:10.1126/science.1134929 (2007).-   14 Xiao, M. et al. Direct determination of haplotypes from single    DNA molecules. Nature methods 6, 199-201, doi:10.1038/nmeth.1301    (2009).-   15 Li, X. et al. Controlled fabrication of fluorescent barcode    nanorods. ACS nano 4, 4350-4360, doi:10.1021/nn9017137 (2010).-   16 Levsky, J. M., Shenoy, S. M., Pezo, R. C. & Singer, R. H.    Single-cell gene expression profiling. Science 297, 836-840,    doi:10.1126/science.1072241 (2002).-   17 Seeman, N. C. Nucleic acid junctions and lattices. Journal of    Theoretical Biology 99, 237-247 (1982).-   18 Aldaye, F. A., Palmer, A. L. & Sleiman, H. F. Assembling    materials with DNA as the guide. Science 321, 1795-1799,    doi:10.1126/science.1154533 (2008).-   19 Lin, C., Liu, Y. & Yan, H. Designer DNA nanoarchitectures.    Biochemistry 48, 1663-1674, doi:10.1021/bi802324w (2009).-   20 Nangreave, J., Han, D., Liu, Y. & Yan, H. DNA origami: a history    and current perspective. Current opinion in chemical biology 14,    608-615, doi:10.1016/j.cbpa.2010.06.182 (2010).-   21 Shih, W. M. & Lin, C. Knitting complex weaves with DNA origami.    Current opinion in structural biology 20, 276-282,    doi:10.1016/j.sbi.2010.03.009 (2010).-   22 Tøning, T., Voigt, N. V., Nangreave, J., Yan, H. & Gothelf, K. V.    DNA origami: a quantum leap for self-assembly of complex structures.    Chemical Society Reviews, doi:10.1039/cics15057j (2011).-   23 Rothemund, P. W. K. Folding DNA to create nanoscale shapes and    patterns. Nature 440, 297-302, doi:10.1038/nature04586 (2006).-   24 Douglas, S. M. et al. Self-assembly of DNA into nanoscale    three-dimensional shapes. Nature 459, 414-418,    doi:10.1038/nature08016 (2009).-   25 Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted    and curved nanoscale shapes. Science 325, 725-730.    doi:10.1126/science.1174251 (2009).-   26 Ke, Y. et al. Multilayer DNA origami packed on a square lattice.    Journal of the American Chemical Society 131, 15903-15908,    doi:10.1021/ja906381y (2009).-   27 Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with    a controllable lid. Nature 459, 73-76, doi:10.1038/nature07971    (2009).-   28 Han, D., Pal, S., Liu, Y. & Yan, H. Folding and cutting DNA into    reconfigurable topological nanostructures. Nature nanotechnology 5,    712-717, doi:10.1038/nnano.2010.193 (2010).-   29 Liedl, T., Högberg, B., Tytell, J., Ingber, D. E. & Shih, W. M.    Self-assembly of three-dimensional prestressed tensegrity structures    from DNA. Nature nanotechnology 5, 520-524,    doi:10.1038/nnano.2010.107 (2010).-   30 Han, D. et al. DNA Origami with Complex Curvatures in    Three-Dimensional Space. Science 332, 342-346,    doi:10.1126/science.1202998 (2011).-   31 Jungmann, R. et al. Single-Molecule Kinetics and Super-Resolution    Microscopy by Fluorescence Imaging of Transient Binding on DNA    Origami. Nano letters, doi:10.1021/n1103427w (2010).-   32 Steinhauer, C., Jungmann, R., Sobey, T. L., Simmel, F. C. &    Tinnefeld, P. DNA origami as a nanoscopic ruler for super-resolution    microscopy. Angewandte Chemie (International ed in English) 48,    8870-8873, doi:10.1002/anie.200903308 (2009).-   33 Lund, K. et al. Molecular robots guided by prescriptive    landscapes. Nature 465, 206-210, doi:10.1038/nature09012 (2010).-   34 Pal, S., Deng, Z., Ding, B., Yan, H. & Liu, Y.    DNA-origami-directed self-assembly of discrete silver-nanoparticle    architectures. Angewandte Chemie (International ed in English) 49,    2700-2704, doi:10.1002/anie.201000330 (2010).-   35 Bui, H. et al. Programmable Periodicity of Quantum Dot Arrays    with DNA Origami Nanotubes. Nano letters 10, 3367-3372,    doi:10.1021/n1101079u (2010).-   36 Liu, W., Zhong, H., Wang, R. & Seeman, N. C. Crystalline    two-dimensional DNA-origami arrays. Angewandte Chemie (International    ed in English) 50, 264-267, doi:10.1002/anie.201005911 (2011).-   37 Woo, S. & Rothemund, P. W. K. Programmable molecular recognition    based on the geometry of DNA nanostructures. Nature Chemistry 3,    620-627, doi:10.1038/nchem.1070 (2011).-   38 Douglas, S. M., Chou, J. J. & Shih, W. M. DNA-nanotube-induced    alignment of membrane proteins for NMR structure determination.    Proceedings of the National Academy of Sciences of the United States    of America 104, 6644-6648, doi:10.1073/pnas.0700930104 (2007).-   39 Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen    scavenging system for improvement of dye stability in    single-molecule fluorescence experiments. Biophysical journal 94,    1826-1835, doi:10.1529/biophysj.107.117689 (2008).-   40 Hell, S. W. Far-field optical manoscopy. Science 316, 1153-1158,    doi:10.1126/science.1137395 (2007).-   41 Hell, S. W. Microscopy and its focal switch. Nature methods 6,    24-32, doi:10.1038/nmeth.1291 (2009).-   42 Huang, B., Babcock, H. & Zhuang, X. Breaking the diffraction    barrier: super-resolution imaging of cells. Cell 143, 1047-1058,    doi:10.1016/j.cell.2010.12.002 (2010).-   43 Vogelsang, J. et al. Make them blink: probes for super-resolution    microscopy. Chemphyschem 11, 2475-2490, doi:10.1002/cphc.201000189    (2010).-   44 Walter, N. G., Huang, C. Y., Manzo, A. J. & Sobhy, M. A.    Do-it-yourself guide: how to use the modern single-molecule toolkit.    Nature Methods 5, 475-489, doi:10.1038/nmeth.1215 (2008).-   45 Hell, S. W. & Wichmann, J. Breaking the Diffraction Resolution    Limit by Stimulated-Emission - Stimulated-Emission-Depletion    Fluorescence Microscopy. Opt Lett 19, 780-782 (1994).-   46 Heilemann, M. et al. Subdiffraction-resolution fluorescence    imaging with conventional fluorescent probes. Angew Chem Int Ed Engl    47, 6172-6176, doi:10.1002/anie.200802376 (2008).-   47 Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging    by stochastic optical reconstruction microscopy (STORM). Nat Methods    3, 793-795 (2006).-   48 Betzig, E. et al. Imaging intracellular fluorescent proteins at    nanometer resolution. Science 313, 1642-1645 (2006).-   49 Steinhauer, C., Forthmann, C., Vogelsang, J. & Tinnefeld, P.    Superresolution

Microscopy on the Basis of Engineered Dark States, Journal of theAmerican Chemical Society 130, 16840-16841, doi:Doi 10.1021/Ja806590m(2008).

-   50 Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction    imaging by accumulated binding of diffusing probes. Proceedings of    the National Academy of Sciences of the United States of America    103, 18911-18916 (2006).-   51 Yildiz, A. et al. Myosin V walks hand-over-hand: single    fluorophore imaging with 1.5-nm localization. Science 300,    2061-2065, doi:10.1126/science.1084398 (2003).-   52 Yildiz, A., Tomishige, M., Vale, R. D. & Selvin, P. R. Kinesin    walks hand-over-hand. Science 303, 676-678,    doi:10.1126/science.1093753 (2004).-   53 Gautier, A. et al. An engineered protein tag for multiprotein    labeling in living cells. Chem Biol 15, 128-136,    doi:10.1016/j.chembio1.2008.01.007 (2008).-   54 Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast,    three-dimensional super-resolution imaging of live cells. Nature    methods 8, 499-508, doi:10.1038/nmeth.1605 (2011).-   55 Keppler, A. et al. A general method for the covalent labeling of    fusion proteins with small molecules in vivo. Nature biotechnology    21, 86-89, doi:10.1038/nbt765 (2003).-   56 Klein, T. et al. Live-cell dSTORM with SNAP-tag fusion proteins.    Nature methods 8, 7-9, doi:10.1038/nmeth0111-7b (2011).-   57 Cunin, F. et al. Biomolecular screening with encoded    porous-silicon photonic crystals. Nature materials 1, 39-41,    doi:10.1038/nmat702 (2002).-   58 Bellot, G., Mcclintock, M. A., Lin, C. & Shih, W. M. Recovery of    intact DNA nanostructures after agarose gel-based separation. Nature    methods 8, 192-194, doi:10.1038/nmeth0311-192 (2011).-   59 Rajendran, A., Endo, M., Katsuda, Y., Hidaka, K. & Sugiyama, H.    Photo-cross-linking-assisted thermal stability of DNA origami    structures and its application for higher-temperature self-assembly.    Journal of the American Chemical Society 133, 14488-14491,    doi:10.1021/ja204546h (2011).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the subject matter described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of detecting a target comprising:contacting a sample with a nucleic acid barcode probe, and determiningwhether the nucleic acid barcode probe binds to one or more componentsin the sample, wherein binding of the nucleic acid barcode probe to oneor more components of the sample indicates presence of a target in thesample, and wherein the nucleic acid barcode probe comprises a nucleicacid nanostructure comprising a target binding moiety and at least twofluorescently labeled regions.
 2. The method of claim 1, furthercomprising identifying the target based on the color and/or orientationof the fluorescently labeled regions of the nucleic acid barcode probebound to one or more components of the sample.
 3. The method of claim 1,wherein whether the nucleic acid barcode probe binds to one or morecomponents in the sample comprises contacting the nucleic acid barcodeprobe with soluble, transiently binding fluorophore-bearingoligonucleotides.
 4. The method of claim 1, wherein the nucleic acidbarcode probe comprises stably bound fluorophore-bearingoligonucleotides.
 5. A method of detecting a target comprisingcontacting the target with a nucleic acid barcode probe, underconditions sufficient for the target to bind to the nucleic acid barcodeprobe; separating the target from material that is not bound to thetarget; and detecting the presence of the nucleic acid barcode probebound to the target, wherein the nucleic acid barcode probe comprises anucleic acid nanostructure comprising a target binding moiety and atleast two fluorescently labeled regions. 6-8. (canceled)
 9. Acomposition comprising a sample, and a nucleic acid barcode probecomprising a nucleic acid nanostructure having at least twofluorescently-labeled regions, wherein: the locations of each of thefluorescently-labeled regions on the nucleic acid nanostructure are suchthat a pattern of the fluorescent labels is determinable based on theemission of visible light by the fluorescently-labeled regions.
 10. Thecomposition of claim 9, wherein the nucleic acid nanostructure comprisesa scaffold strand and a plurality of staple strands.
 11. The compositionof claim 9, wherein the fluorescently-labeled regions comprisefluorophore-labeled staple strands.
 12. The composition of claim 11,wherein the fluorophore-labeled staple strands comprise (a) a stapledomain hybridized to a scaffold strand and (b) a handle domain. 13.(canceled)
 14. The composition of claim 9, wherein the nucleic acidnanostructure is a DNA nanostructure.
 15. The composition of claim 14,wherein the DNA nanostructure is a nanotube.
 16. The composition ofclaim 9, wherein the nucleic acid nanostructure comprises at least threefluorescently-labeled regions or at least four fluorescently-labeledregions.
 17. The composition of claim 16, wherein orientation of the atleast three or at least four fluorescently labeled regions isdeterminable based on asymmetric spacing of the fluorescently-labeledregions.
 18. The composition of claim 9, wherein each of thefluorescently-labeled regions has a center that is located at leastabout 50 nm or at least about 100 nm from the centers of the otherfluorescently-labeled regions.
 19. The composition of claim 9, whereineach of the fluorescently-labeled regions has a center that is locatedat least 200 nm from the centers of the other fluorescently-labeledregions. 20-24. (canceled)
 25. A method of identifying a target nucleicacid comprising: contacting the target nucleic acid with a nucleic acidbarcode probe of claim 9, under conditions such that the target nucleicacid binds to the nucleic acid barcode probe; separating the targetnucleic acid from material that is not bound to the target nucleic acid;and detecting the presence of the nucleic acid barcode probe bound tothe target nucleic acid.
 26. A method of identifying a target protein orcell comprising: contacting the target protein or cell with a nucleicacid barcode probe of claim 9, under conditions such that the targetprotein or cell binds to the nucleic acid barcode probe; separating thetarget protein or cell from material that is not bound to the targetprotein or cell; and detecting the presence of the nucleic acid barcodeprobe bound to the target protein or cell.
 27. A method of identifying atarget protein or cell comprising: contacting the target protein or cellwith a nucleic acid barcode probe of claim 9, under conditions such thatthe target protein or cell binds to the nucleic acid barcode probe;separating the target protein or cell from material that is not bound tothe target protein or cell; and detecting the presence of the nucleicacid barcode probe bound to the target protein or cell.
 28. A method ofdetecting a target comprising: contacting a sample with a nucleic acidbarcode probe of claim 9, and determining whether the nucleic acidbarcode probe binds to one or more components in the sample, whereinbinding of the nucleic acid barcode probe to one or more components inthe sample indicates presence of a target in the sample. 29-32.(canceled)
 33. A population of nucleic acid barcode probes, wherein eachprobe comprises a nucleic acid nanostructure having at least twofluorescently-labeled regions and a target binding moiety, wherein thelocations of each of the fluorescently-labeled regions on the nucleicacid nanostructure are such that a pattern of the fluorescent labels ofthe nucleic acid barcode probe and the identity of the target bindingmoiety is determinable based on the emission of visible light by thefluorescently-labeled regions.
 34. (canceled)